Human babesiosis is a tick-borne malaria-like illness with a worldwide distribution and endemic in the United States. The disease joined the list of CDC-nationally notifiable diseases in 2011 due to the increasing number of reported cases. The majority of clinical cases in the US have been linked to Babesia microti whereas Babesia duncani cases have been reported primarily from Washington, Oregon, and California. However, due to the lack of specific diagnostic assays and because immune competent individuals can be asymptomatic, the true incidence of babesiosis cases in the US and worldwide is most likely underestimated. There is no vaccine against human babesiosis and current therapies are not effective, require high doses and some of them are associated with major side effects. Even with these drugs and other supportive measures, the case fatality rate exceeds 20% in susceptible hosts. During the past 8 years, we gained significant understanding of the biology, pathogenesis and drug susceptibility of Babesia parasites that infect humans: (1) we completed the first genomic and transcriptomic analyses and annotations of B. microti and B. duncani; (2) probed the genomic diversity of B. microti in human blood and ticks; (3) demonstrated that B. microti and B. duncani are inherently tolerant to clinically recommended therapies; (4) discovered a combination therapy that results in radical cure of B. microti infection in mice; (5) completed the first immunoproteomic analysis of B. microti, identified the main immunodominant antigens of this parasite, and developed the first diagnostic assay for detection of active B. microti infection; and (6) discovered a vesicular-based secretion system used by the parasite to export some of its proteins to the host. Of particular relevance to the current RFA, we used immunoproteomic and proteomic approaches to identify secreted and surface-displayed proteins of B. microti and B. duncani, and for more than 30 B. microti antigens determine their serological profile in sera from infected mice and humans. The main hypothesis of this proposal is that some of the exported antigens of B. microti and B. duncani could serve as vaccines to illicit antibodies to block Babesia invasion of host erythrocytes or to facilitate elimination of Babesia- infected erythrocytes by host macrophages. The primary goals of this application are to further characterize the immunogenicity profile of these secreted antigens, and to conduct vaccination studies in mice to identify those antigens that could be developed as vaccines against Babesia microti and B. duncani infections. In Aim 1, we will produce functional recombinant forms of secreted and surface displayed proteins of B. microti and B. duncani and use them to determine the serological profile and kinetics of the humoral immune responses using mouse and human sera. In Aim 2. We will conduct cell biological studies to determine the secretion and cellular distribution of the candidate exported antigens during the intraerythrocytic life cycle of these parasites. In Aim 3, we will conduct immunization studies in mouse models of B. microti and B. duncani infections to identify those antigens that are effective vaccines when administered in human-compatible adjuvants.